Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues
Quantitative polymerase chain reaction (qPCR) has emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products after xenotransplantation into immunodeficient mice. A particular challenge in bioanalytical qPCR studies is that the different tis...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-07-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/12/13/1788 |
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| author | Hannes M. Schröder Elke Niebergall-Roth Alexandra Norrick Jasmina Esterlechner Christoph Ganss Markus H. Frank Mark A. Kluth |
| author_facet | Hannes M. Schröder Elke Niebergall-Roth Alexandra Norrick Jasmina Esterlechner Christoph Ganss Markus H. Frank Mark A. Kluth |
| author_sort | Hannes M. Schröder |
| collection | DOAJ |
| description | Quantitative polymerase chain reaction (qPCR) has emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products after xenotransplantation into immunodeficient mice. A particular challenge in bioanalytical qPCR studies is that the different tissues of the host organism can affect amplification efficiency and amplicon detection to varying degrees, and ignoring these matrix effects can easily cause a significant underestimation of the true number of target cells in a sample. Here, we describe the development and drug regulatory-compliant validation of a TaqMan<sup>®</sup> qPCR assay for the quantification of mesenchymal stromal cells in the range of 125 to 20,000 cells/200 µL lysate via the amplification of a human-specific, highly repetitive α-satellite DNA sequence of the chromosome 17 centromere region HSSATA17. An assessment of matrix effects in 14 different mouse tissues and blood revealed a wide range of spike recovery rates across the different tissue types, from 11 to 174%. Based on these observations, we propose performing systematic spike-and-recovery experiments during assay validation and correcting for the effects of the different tissue matrices on cell quantification in subsequent bioanalytical studies by multiplying the back-calculated cell number by tissue-specific factors derived from the inverse of the validated percent recovery rate. |
| first_indexed | 2024-03-11T01:45:01Z |
| format | Article |
| id | doaj.art-408e81adead745d68d6fb5d66f0f37d6 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-11T01:45:01Z |
| publishDate | 2023-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-408e81adead745d68d6fb5d66f0f37d62023-11-18T16:20:04ZengMDPI AGCells2073-44092023-07-011213178810.3390/cells12131788Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ TissuesHannes M. Schröder0Elke Niebergall-Roth1Alexandra Norrick2Jasmina Esterlechner3Christoph Ganss4Markus H. Frank5Mark A. Kluth6RHEACELL GmbH & Co. KG, 69120 Heidelberg, GermanyRHEACELL GmbH & Co. KG, 69120 Heidelberg, GermanyRHEACELL GmbH & Co. KG, 69120 Heidelberg, GermanyRHEACELL GmbH & Co. KG, 69120 Heidelberg, GermanyRHEACELL GmbH & Co. KG, 69120 Heidelberg, GermanyDepartment of Dermatology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USARHEACELL GmbH & Co. KG, 69120 Heidelberg, GermanyQuantitative polymerase chain reaction (qPCR) has emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products after xenotransplantation into immunodeficient mice. A particular challenge in bioanalytical qPCR studies is that the different tissues of the host organism can affect amplification efficiency and amplicon detection to varying degrees, and ignoring these matrix effects can easily cause a significant underestimation of the true number of target cells in a sample. Here, we describe the development and drug regulatory-compliant validation of a TaqMan<sup>®</sup> qPCR assay for the quantification of mesenchymal stromal cells in the range of 125 to 20,000 cells/200 µL lysate via the amplification of a human-specific, highly repetitive α-satellite DNA sequence of the chromosome 17 centromere region HSSATA17. An assessment of matrix effects in 14 different mouse tissues and blood revealed a wide range of spike recovery rates across the different tissue types, from 11 to 174%. Based on these observations, we propose performing systematic spike-and-recovery experiments during assay validation and correcting for the effects of the different tissue matrices on cell quantification in subsequent bioanalytical studies by multiplying the back-calculated cell number by tissue-specific factors derived from the inverse of the validated percent recovery rate.https://www.mdpi.com/2073-4409/12/13/1788ABCB5biodistributioncell therapyHSSATA17 sequencemesenchymal stromal cellsquantitative polymerase chain reaction |
| spellingShingle | Hannes M. Schröder Elke Niebergall-Roth Alexandra Norrick Jasmina Esterlechner Christoph Ganss Markus H. Frank Mark A. Kluth Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues Cells ABCB5 biodistribution cell therapy HSSATA17 sequence mesenchymal stromal cells quantitative polymerase chain reaction |
| title | Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues |
| title_full | Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues |
| title_fullStr | Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues |
| title_full_unstemmed | Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues |
| title_short | Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues |
| title_sort | drug regulatory compliant validation of a qpcr assay for bioanalysis studies of a cell therapy product with a special focus on matrix interferences in a wide range of organ tissues |
| topic | ABCB5 biodistribution cell therapy HSSATA17 sequence mesenchymal stromal cells quantitative polymerase chain reaction |
| url | https://www.mdpi.com/2073-4409/12/13/1788 |
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